The present invention relates to gene therapy. In particular it relates to vectors for use in the preparation of recombinant lentiviruses and the use of replication-deficient lentiviral vectors to deliver a therapeutic gene to a target tissue of a subject. Suitable therapeutic genes include genes that encode clotting factors, such as factor VIII or factor IX, to treat a blood clotting disease such as hemophilia. The invention further relates to enhancers and promoters useful for tissue-specific gene therapy.
Gene therapy generally relates to the delivery of one or more heterologous genes to a subject in order to treat a disease. Hemophilia is a genetic disease caused by a deficiency of a blood clotting factor. There are two types of X-linked bleeding disorders, hemophilia A and hemophilia B. In some cases of von Willebrand disease, the most common bleeding disorder, deficient levels of vWF result in low levels of factor VIII, mimicking hemophilia A. Hemophilia A affects about 17,000 people in the US and is caused by a deficiency in factor VIII. The incidence of hemophilia B is 1 out of 34,500 men, and it is caused by a deficiency in factor IX. Each of these diseases is an excellent theoretical candidate for gene therapy, as each has a reasonably simple molecular pathology and should be remediable by the delivery of a single gene.
Successful gene therapy for hemophilia requires both tissue specific expression, to avoid a counterproductive immune response, and sufficiently high levels of expression to generate a therapeutic response. Gene therapy directed at quiescent cells of the liver presents an additional challenge, as Park et al. teach that stable and efficient transduction of liver cells with a lentiviral vector requires cell proliferation (Park et al., 2000, Nature Genetics 24:49-52). Park et al. further teach that the injection of doses of the lentiviral vector sufficiently high to reach therapeutic levels of transgene expression in the liver produces a very high liver toxicity and a high mortality (Park et al., 2000, Blood 96(3):1173-1176).
There remains a need for successful gene therapy of quiescent cells that results in therapeutically acceptable cell transduction and that produces a therapeutic amount of protein without toxicity. There is a particular need for a safe and efficient gene therapy for hemophilia.
The invention is based on the surprising discovery that replication deficient lentiviral vectors can be used to achieve stable genetic modification of cells in vivo without vector-mediated toxicity and in the absence of target cell proliferation. The invention thus provides vectors for transgene delivery as well as methods for gene therapy using such vectors. The invention further provides promoters and enhancers useful for optimizing tissue specific transgene delivery.
The invention provides a lentiviral producer cell comprising a first nucleotide sequence comprising a gag, a pol, or gag and pol genes; a second nucleotide sequence comprising a heterologous env gene; and a third nucleotide sequence comprising a lentiviral transfer vector that comprises a gene that encodes a blood clotting factor operably linked to an expression control sequence; wherein the producer cell lacks a functional tat gene. In preferred embodiments, the blood clotting factor comprises human factor IX (hFIX) or human factor VIII (hFVIII). In one embodiment, the first, second and third nucleotide sequences are stably integrated into the genome of the lentiviral producer cell. Preferably, the lentivirus is a human immunodeficiency virus (HIV), such as HIV-1. In a preferred embodiment, the producer cell further comprises a fourth nucleotide sequence that comprises a rev gene, and/or lacks functional accessory genes vif, vpr, vpu, vpx and nef, or a combination thereof.
Typically, the expression control sequence comprises a liver specific promoter, such as mouse transthyretin (mTTR) promoter, human alpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, a human factor VIII endogenous promoter, and/or mouse albumin promoter, and/or a liver specific binding site for transcription, such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, or a combination thereof Preferably, the expression control sequence comprises an Enh1 enhancer (SEQ ID NO: 10) and an mTTR promoter. Also preferred is an expression control sequence comprising an xcex1-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).
The invention additionally provides set of lentiviral vectors for use in a third generation lentiviral packaging system and for production of lentiviral producer cells and recombination lentiviruses. The invention provides a lentiviral transfer vector that comprises an expression control sequence operably linked to a transgene, wherein the expression control sequence comprises a novel enhancer element, Enh1 (SEQ ID NO: 10), or a novel combination of enhancer and promoter elements, such as L-F8 or Enh1 and mTTR. The transgene is preferably a therapeutic transgene. A preferred transgene is one that encodes a blood clotting factor, such as human Factor VIII or human Factor IX. Such transgenes encoding blood clotting factors can be used in the treatment of bleeding disorders such as hemophilia. Also provided is a method of producing a lentiviral producer cell comprising transforming a cell with the set of vectors of the invention. The invention further provides a method of producing a recombinant lentivirus, and a method of delivering a transgene to a cell.